Cryopreserved phhs

Cryopreserved PHHs (454543; Corning) were thawed with cryopreserved hepatocyte plating medium and cryopreserved hepatocyte recovery medium (Thermo Fisher Scientific) according to manufacturer instructions in a Matrigel-coated dish at 2 × 10⁵ cells/cm². The medium was replaced with fresh hepatocyte culture medium (cc-3199; Lonza Group AG, Basel, Switzerland) at 4 to 6 h after thawing. HepG2 cells (ATCC) were cultured in Dulbecco Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Corning) and 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific). To evaluate CYP3A4-mediated hepatotoxicity, DM-differentiated organoids were treated with troglitazone (50 μM) and propionate (1 μM), an SCFA mixture (acetate:propionate:butyrate = 1:1:1), or CYP3A4 inhibitor ketoconazole (1 μM) 3 days after differentiation for another 6 days. PHHs and HepG2 cells were seeded in 96-well plates, and the following day, a troglitazone and SCFA mixture was treated daily for 3 days. Toxicity was evaluated using EZ-cytox (Dogenbio, Seoul, Korea) following the manufacturer’s instructions. The absorbance of the medium was measured using a Spectra Max M3 microplate reader (Molecular Devices, San Jose, CA, USA).